An engineered Fc variant of an IgG eliminates all immune effector functions via structural perturbations

The Fc variant of IgG2, designated as IgG2cr, was engineered with V234A/G237A /P238S/H268A/V309L/ A330S/P331S substitutions to eliminate affinity for Fcy receptors and Clq complement protein and consequently, immune effector functions. IgGar was compared to other previously well-characterized Fc 'muted' variants, including aglycosylated IgGl, IgG2m4 (H2680Jv309L1A330S/P331S, changes to IgG4), and IgG4 ProAlaAla (S228P/L234A1L235A) in its capacity to bind FcyRs and activate various immunestimulatory responses. In contrast to the previously characterized muted Fc variants, which retain selective FcyR binding and effector functions, IgG26 shows no detectable binding to the Fcy receptors in affinity and avidity measurements, nor any detectable antibody-dependent cytotoxicity, phagocytosis, complement activity, or Fc-mediated cytokine release. Moreover, IgG2a shows minimal immunogenic potential by T-cell epitope analysis. The circulating half-life of IgG2a in monkeys is extended relative to IgG1 and IgG2, in spite of similar in vitro binding to recombinant FcRn. The three-dimensional structure of the Fc, needed for assessing the basis for the absence of effector function, was compared with that of IgG2 revealing a number of conformational differences near the hinge region of the CH2 domain that result from the amino acid substitutions. Modeling reveals that at least one of the key interactions with FcyRs is disrupted by a conformational change that reorients P329 to a position that prevents it from interacting with conserved W90 and W113 residues of the FcyRs. Inspection of the structure also indicated significant changes to the conformations of D270 and P329 in the CH2 domain that could negatively impact Cl q binding. Thus, structural perturbations of the Fc provide a rationale for the loss of function. In toto, these properties of IgG2a suggest that it is a superior alternative to previously described IgG variants of minimal effector function, for future therapeutic applications of non-immunostimulatory mAb and Fcfusion platforms. (C) 2013 Elsevier Inc. All rights reserved.